Mutant Cytochrome Protein and Use Thereof

ABSTRACT

A mutant cytochrome protein originated from a cytochrome protein having three heme-binding domains, which mutant cytochrome protein lacks the first heme-binding domain and the second heme-binding domain as counted from the N-terminus, is provided. The mutant cytochrome protein may lack a region(s) containing the first and second heme-binding domains.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled “SequenceListing.txt,” created on or about Jul. 2, 2018 with a file size of about 56 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a mutant cytochrome protein capable of electron transfer even at a low electric potential. The mutant cytochrome protein according to one embodiment of the present invention can be suitably used for biosensors and the like, and is useful in the fields of biochemistry, medicine, and the like.

BACKGROUND ART

Cytochrome protein is known as an electron acceptor protein, and used as an electrochemical sensor by formation of a complex with a catalytic subunit of an oxidoreductase.

For example, WO 2005/023111 discloses use of a complex of the catalytic subunit (α-subunit) and the cytochrome c subunit β-subunit) of glucose dehydrogenase derived from a Burkholderia microorganism as a biosensor. Such a biosensor is used for measurement of the glucose concentration or the like in a biological sample. For more accurate measurement, the measurement sensitivity needs to be increased by, for example, modification of the protein.

The mutants of electrochemical sensor proteins that have been studied so far are mainly mutants of a catalytic subunit, and mutants of a cytochrome protein have been hardly studied.

SUMMARY OF THE INVENTION

In measurement of a target substance using a biosensor that monitors the oxidation-reduction current, it is preferred to reduce the oxidation-reduction potential to within the range in which electron transfer between an enzyme or an enzyme complex and an electrode can be monitored, from the viewpoint of reduction of influences of reducing substances contained in the sample.

However, in cases where the oxidation-reduction current is monitored using a conventional naturally occurring cytochrome protein as an electron acceptor protein, application of a high electric potential to the electrode is required. For example, in Example 7 of WO 2005/023111, an electric potential of +350 nM is applied with respect to a silver-silver chloride electrode. At this electric potential, there is a concern that the measured value may be positively biased due to oxidation of reducing substances such as ascorbic acid and acetaminophen.

In view of such a problem, one aspect of the present invention is to provide a mutant electron acceptor subunit protein that can be used for a biosensor capable of measuring a target substance by application of a low electric potential.

The present inventors prepared a mutant cytochrome c protein lacking a region containing the first heme-binding domain and the second heme-binding domain as counted from the amino-terminus. The present inventors then carried out evaluation of a biosensor using this protein. Surprisingly, as a result, the present inventors found that a mutant cytochrome c protein lacking a region(s) containing the first and second heme-binding domains is capable of accepting an electron from a catalytic subunit irrespective of the fact that the protein has only one heme-binding domain (third heme-binding domain), allowing an electric current to flow depending on the target substance even at an electric potential lower than those in conventional cases, thereby completed the present invention.

One aspect of the present invention is to provide a mutant cytochrome protein originated from a cytochrome protein having three heme-binding domains, the mutant cytochrome protein lacking the first heme-binding domain and the second heme-binding domain as counted from the N-terminus.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein said mutant cytochrome protein lacks a region(s) comprising the first and second heme-binding domains.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein the region comprising the first and second heme-binding domains corresponds to the region of amino acids from 43 to 195 of SEQ ID NO:4.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein the cytochrome protein is a cytochrome c protein.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein the cytochrome c protein having three heme-binding domains is that of a Burkholderia microorganism.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein the cytochrome c protein having three heme-binding domains is that of Burkholderia cepacia.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein the original cytochrome protein having three heme-binding domains comprises an amino acid sequence which is at least 60% identical to SEQ ID NO:4.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein said mutant cytochrome protein consists of the amino acid sequence selected from the group consisting of:

a) the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4, b) the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, c) an amino acid sequence which is the same as the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4 except that one or several amino acids are substituted, deleted, inserted, and/or added with the proviso that the CXXCH motif of amino acids 334 to 338 is maintained, and d) an amino acid sequence which is the same as the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4 except that one or several amino acids are substituted, deleted, inserted, and/or added with the proviso that the CXXCH motif of amino acids 334 to 338 is maintained.

Another aspect of the present invention is to provide the mutant cytochrome protein as described above, wherein said mutant cytochrome protein consists of the amino acid sequence selected from the group consisting of:

a) the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4, b) the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, c) an amino acid sequence which is at least 90% identical to the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4 with the proviso that the CXXCH motif of amino acids 334 to 338 is maintained, and d) an amino acid sequence which is at least 90% identical to the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4 with the proviso that the CXXCH motif of amino acids 334 to 338 is maintained.

Another aspect of the present invention is to provide a DNA encoding the mutant cytochrome protein as described above.

Another aspect of the present invention is to provide a recombinant vector comprising the DNA as described above.

Another aspect of the present invention is to provide a cell transformed with the recombinant vector as described above.

Another aspect of the present invention is to provide an oxidoreductase-cytochrome complex comprising the mutant cytochrome protein as described above and a catalytic subunit protein of an oxidoreductase.

Another aspect of the present invention is to provide the oxidoreductase-cytochrome complex as described above, wherein the oxidoreductase is glucose dehydrogenase.

Another aspect of the present invention is to provide a biosensor comprising an enzyme electrode comprising the oxidoreductase-cytochrome complex as described above.

Another aspect of the present invention is to provide a method for measuring a measurement target substance, comprising adding a sample to the biosensor as described above, applying an electric potential, measuring the response current, and then calculating the concentration of the measurement target substance contained in the sample based on the response current. In particular, the method serves to measure the concentration of the target substance in a sample.

Another aspect of the present invention is to provide the method as described above, wherein an electric potential of 0 to +300 mV is applied to the enzyme electrode with respect to a silver-silver chloride electrode.

The present invention enables construction of an electrochemical biosensor that operates at an electric potential lower than those in conventional cases (for example, by application of 0 to +300 mV, 0 to +150 mV, or 0 to +100 mV with respect to a silver-silver chloride electrode), thereby allowing accurate measurement of a target substance such as glucose by suppression of interference by reducing substances in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the results of chronoamperometry using a glucose sensor containing a glucose dehydrogenase (GDH) complex having a wild-type β-subunit or a glucose sensor containing a GDH complex having an N-terminal region-deleted β-subunit (the electric potential applied was 0, +100, or +400 mV).

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Embodiments of the present invention are described below in detail.

The mutant cytochrome protein according to one embodiment of the present invention is a mutant of a cytochrome protein having three heme-binding domains, which mutant lacks the first heme-binding domain and the second heme-binding domain as counted from the N-terminus. The protein originates from a cytochrome protein having three heme-binding domains. As used herein, a protein which “originates” refers to a protein which is derived from the parent/original cytochrome protein to provide the mutated cytochrome protein, e.g. by mutation. The original cytochrome protein contains the three heme-binding domains.

Cytochrome protein has three heme-binding domains, and the first, second, and third heme-binding domains as counted from the N-terminus are referred to as first, second, and third heme-binding domains, respectively, in the present specification. A heme-binding domain is generally represented as CXXCH (SEQ ID NO:5) (wherein X represents an arbitrary amino acid), and the mutant cytochrome protein according to one embodiment of the present invention is obtained by modifying a wild type cytochrome protein such that it lacks the first and second heme-binding domains.

Here, “modified such that it lacks the first and second heme-binding domains” means that the protein lacks the first and second CXXCH motifs.

The lack of the CXXCH motifs includes lack of a region(s) containing the CXXCH motifs. The protein may lack a region containing the first heme-binding domain and a region containing the second heme-binding domain separately, or may lack a region containing the first and second heme-binding domains.

The cytochrome protein may be a cytochrome c protein. Examples of the cytochrome c protein include the cytochrome c protein of Burkholderia cepacia represented by SEQ ID NO:4 (β-subunit of glucose dehydrogenase).

The cytochrome c protein of Burkholderia cepacia is described below as a representative example.

In SEQ ID NO:4, the first heme-binding domain (amino acids 43 to 47), the second heme-binding domain (amino acids 191 to 195), and the third heme-binding domain (amino acids 334 to 338) are present. The lack of the first heme-binding domain and the second heme-binding domain may be achieved by lacking (e.g. by deletion of) of a region containing the first and second heme-binding domains (amino acids 43 to 195).

As long as the third heme-binding domain is maintained and allows the protein to function as a mutant cytochrome c protein, that is, as long as the electron transfer ability is maintained, the lack of the region containing the first and second heme-binding domains (amino acids 43 to 195) may be lack of at least this region, and may be lack of not only this region, but also amino acids before and after this region in SEQ ID NO:4. For example, the protein may lack the region from the N-terminus to the amino acid at position 195 of SEQ ID NO:4. An example of the mutant cytochrome c protein lacking a region containing the first and second heme-binding domains is the mutant cytochrome c protein composed of amino acid positions 314 to 425 or 330 to 425 of SEQ ID NO:4. However, in these sequences, an arbitrary tag sequence(s) and/or signal sequence(s) (for example, amino acids 1 to 27 of SEQ ID NO:4) may be added to the N-terminal side and/or the C-terminal side.

The mutant cytochrome protein according to one embodiment of the present invention is not limited to the amino acid sequence consisting of amino acids 314 to 425 of SEQ ID NO:4 or the amino acid sequence consisting of amino acids 330 to 425 of SEQ ID NO:4 itself, and may be a sequence which is the same as this sequence except that one or several amino acids in a region(s) other than the third heme-binding domain (amino acids 334 to 338) are substituted, deleted, added, and/or inserted, as long as the electron transfer ability is maintained. The term “one or several” herein means, for example, 1 to 20, 1 to 10, 1 to 8, 1 to 5, or 1 to 3, i.e. 1, 2 or 3. The substitution may be conservative substitution. “Conservative substitution” means substitution between amino acids having similar properties, such as substitution between acidic amino acids, substitution between neutral amino acids, or substitution between basic amino acids. To the N-terminal side of the amino acid sequence consisting of amino acids 314 to 425 of SEQ ID NO:4, part of the C-terminal sequence of the amino acid sequence consisting of amino acids 196 to 313 of SEQ ID NO:4 may be added. To the N-terminal side of the amino acid sequence consisting of amino acids 330 to 425 of SEQ ID NO:4, part of the C-terminal sequence of the amino acid sequence consisting of amino acids 196 to 329 of SEQ ID NO:4 may be added

As long as the third heme-binding domain (amino acids 334 to 338) is maintained and the electron transfer ability is maintained, the mutant cytochrome c protein may be a protein consisting of an amino acid sequence with a sequence identity of not less than 80%, not less than 90%, or not less than 95% to the amino acid sequence consisting of amino acids 314 to 425 of SEQ ID NO:4 or the amino acid sequence consisting of amino acids 330 to 425 of SEQ ID NO:4.

SEQ ID NO:4 is the amino acid sequence of the GDH β-subunit of the Burkholderia cepacia KS1 strain. The KS1 strain has been deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (at present, National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) under accession No. FERM BP-7306 as of Sep. 25, 2000.

The cytochrome c protein before the introduction of the modification (original cytochrome c protein) is not limited to the cytochrome c protein of Burkholderia cepacia represented by SEQ ID NO:4 as long as it has the first, second, and third heme-binding domains. For example, a homologue of SEQ ID NO:4 may be used, and the homologue may have an identity of not less than 60%, not less than 80%, or not less than 90% or 95% to SEQ ID NO:4.

The amino acid sequence of the cytochrome c protein having a sequence identity of not less than 60% to SEQ ID NO:4 may be a cytochrome c protein of the genus Burkholderia. Examples of such a cytochrome c protein include the cytochrome protein of the Burkholderia cepacia J2315 strain (SEQ ID NO:6), the cytochrome c protein of Burkholderia cenocepacia (SEQ ID NO:7), the cytochrome c protein of Burkholderia multivorans (SEQ ID NO:8), the cytochrome c protein of Burkholderia ubonensis (SEQ ID NO:9), the cytochrome c protein of Burkholderia stagnalis (SEQ ID NO:10), and the cytochrome c protein of Burkholderia thailandensis (SEQ ID NO:11).

The cytochrome protein may be a cytochrome protein of a genus other than Burkholderia, such as a cytochrome protein of Ralstonia solanacearum (SEQ ID NO:12) or a cytochrome protein of Ralstonia pickettii (SEQ ID NO:13). The cytochrome protein is not limited to these, and may be a cytochrome c protein derived from another organism. Examples of the amino acid sequences of cytochrome c proteins having a sequence identity of not less than 60% to SEQ ID NO:4 are shown below with accession numbers for Protein Database of National Center for Biotechnology Information (NCBI). In each of these sequences, three heme-binding domains are present. The mutant cytochrome protein can be obtained by deleting the first heme-binding domain and the second heme-binding domain as counted from (i.e. starting from) the amino-terminus.

WP_006403391.1 MULTISPECIES: cytochrome C [Burkholderia] AAQ06608.1 glucose dehydrogenase beta subunit [Burkholderia cepacia] WP_006396899.1 cytochrome C [Burkholderia multivorans] (SEQ ID NO:8) SAJ95286.1 gluconate 2-dehydrogenase (acceptor) [Burkholderia multivorans] WP_006412653.1 cytochrome C [Burkholderia multivorans] WP_060041792.1 cytochrome C [Burkholderia multivorans] WP_060112921.1 cytochrome C [Burkholderia multivorans] WP_060182288.1 cytochrome C [Burkholderia multivorans] WP_060151834.1 cytochrome C [Burkholderia multivorans] WP_048804658.1 cytochrome C [Burkholderia multivorans] WP_059786407.1 cytochrome C [Burkholderia multivorans] WP_035955019.1 cytochrome C [Burkholderia multivorans] WP_059585013.1 cytochrome C [Burkholderia anthina] WP_006482958.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] (SEQ ID NO:6) WP_048988065.1 cytochrome C [Burkholderia cenocepacia] (SEQ ID NO:7) WP_084751507.1 cytochrome C [Burkholderia cenocepacia] WP_077186512.1 cytochrome C [Burkholderia cenocepacia] WP_069351905.1 cytochrome C [Burkholderia cenocepacia] WP_059783319.1 cytochrome C [Burkholderia sp. NRF60-BP8] WP_060263394.1 cytochrome C [Burkholderia cenocepacia] WP_059836247.1 cytochrome C [Burkholderia sp. MSMB1835] WP_059556637.1 cytochrome C [Burkholderia seminalis] WP_063551853.1 cytochrome C [Burkholderia territorii] WP_060310848.1 cytochrome C [Burkholderia anthina] WP_034187893.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_059789576.1 cytochrome C [Burkholderia sp. MSMB1072] WP_059500343.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_034204695.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_011882359.1 cytochrome C [Burkholderia vietnamiensis] WP_060968838.1 cytochrome C [Burkholderia anthina] WP_077204198.1 cytochrome C [Burkholderia cenocepacia] WP_050012790.1 cytochrome C [Burkholderia cenocepacia] WP_011547563.1 MULTISPECIES: cytochrome C [Burkholderia] WP_014724780.1 MULTISPECIES: cytochrome C [Burkholderia] WP_059453668.1 cytochrome C [Burkholderia vietnamiensis] WP_034195126.1 MULTISPECIES: cytochrome C [Burkholderia] WP_027812584.1 cytochrome C [Burkholderia cenocepacia] WP_059720348.1 cytochrome C [Burkholderia vietnamiensis] WP_059548127.1 cytochrome C [Burkholderia vietnamiensis] WP_044843288.1 cytochrome C [Burkholderia sp. USM B20] WP_059577619.1 cytochrome C [Burkholderia vietnamiensis] WP_059459380.1 cytochrome C [Burkholderia vietnamiensis] WP_059451171.1 cytochrome C [Burkholderia territorii] WP_027780832.1 cytochrome C [Burkholderia cepacia] WP_077217239.1 cytochrome C [Burkholderia cenocepacia] WP_059539736.1 cytochrome C [Burkholderia diffusa] WP_059507572.1 cytochrome C [Burkholderia territorii] AOJ19136.1 cytochrome C [Burkholderia cenocepacia] WP_027806162.1 cytochrome C [Burkholderia cenocepacia] WP_060107812.1 cytochrome C [Burkholderia territorii] WP_059822261.1 cytochrome C [Burkholderia sp. MSMB1826] WP_059464697.1 cytochrome C [Burkholderia diffusa] WP_059240315.1 cytochrome C [Burkholderia cepacia] WP_059734410.1 cytochrome C [Burkholderia vietnamiensis] WP_069227164.1 cytochrome C [Burkholderia diffusa] WP_060118353.1 cytochrome C [Burkholderia territorii] WP_069617959.1 cytochrome C [Burkholderia sp. A2] WP_060347293.1 cytochrome C [Burkholderia territorii] WP_059702517.1 cytochrome C [Burkholderia vietnamiensis] WP_060126789.1 cytochrome C [Burkholderia territorii] WP_069260162.1 cytochrome C [Burkholderia metallica] WP_059978049.1 cytochrome C [Burkholderia territorii] WP_059691288.1 cytochrome C [Burkholderia sp. RF4-BP95] WP_040140035.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_011349243.1 cytochrome C [Burkholderia lata] WP_059533875.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_059547167.1 cytochrome C [Burkholderia latens] WP_057924955.1 cytochrome C [Burkholderia ambifaria] WP_059607236.1 cytochrome C [Burkholderia sp. LA-2-3-30-S1-D2] WP_039351157.1 MULTISPECIES: cytochrome C [Burkholderia] WP_046548033.1 cytochrome C [Burkholderia contaminans] WP_006752013.1 cytochrome C [Burkholderia ambifaria] WP_014899067.1 cytochrome C [Burkholderia cepacia] WP_006756339.1 cytochrome C [Burkholderia ambifaria] WP_059819259.1 cytochrome C [Burkholderia sp. MSMB0856] WP_011354899.1 cytochrome C [Burkholderia lata] WP_039320755.1 cytochrome C [Burkholderia sp. A9] WP_035974222.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_071332428.1 cytochrome C [Burkholderia contaminans] WP_059684703.1 cytochrome C [Burkholderia sp. FL-7-2-10-S1-D7] WP_048252126.1 cytochrome C [Burkholderia cepacia] WP_069251790.1 cytochrome C [Burkholderia lata] WP_011658978.1 cytochrome C [Burkholderia ambifaria] WP_012366204.1 cytochrome C [Burkholderia ambifaria] WP_072438780.1 MULTISPECIES: cytochrome C [Burkholderia] WP_031400525.1 MULTISPECIES: cytochrome C [Burkholderia] WP_034180248.1 cytochrome C [Burkholderia pyrrocinia] WP_043184377.1 cytochrome C [Burkholderia cepacia] WP_059729924.1 cytochrome C [Burkholderia cepacia] WP_059713002.1 cytochrome C [Burkholderia ubonensis] (SEQ ID NO:9) WP_065501791.1 cytochrome C [Burkholderia stabilis] WP_059615074.1 cytochrome C [Burkholderia ubonensis] WP_085037374.1 cytochrome C [Burkholderia sp. CAMPA 1040] WP_060359188.1 cytochrome C [Burkholderia cepacia] WP_060333590.1 cytochrome C [Burkholderia ubonensis] WP_059522548.1 cytochrome C [Burkholderia cepacia] WP_059766841.1 cytochrome C [Burkholderia ubonensis] WP_059637760.1 cytochrome C [Burkholderia ubonensis] WP_040131620.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] WP_060052348.1 cytochrome C [Burkholderia cepacia] WP_021162032.1 MULTISPECIES: glucose dehydrogenase [Burkholderia] WP_059666051.1 cytochrome C [Burkholderia cepacia] WP_060375463.1 cytochrome C [Burkholderia cepacia] WP_060371861.1 cytochrome C [Burkholderia cepacia] WP_059697397.1 cytochrome C [Burkholderia cepacia] WP_047903070.1 cytochrome C [Burkholderia pyrrocinia] WP_027791850.1 cytochrome C [Burkholderia cepacia] WP_059552416.1 cytochrome C [Burkholderia ubonensis] WP_060232513.1 cytochrome C [Burkholderia cepacia] WP_059687063.1 cytochrome C [Burkholderia cepacia] WP_060226065.1 cytochrome C [Burkholderia cepacia] WP_060125713.1 cytochrome C [Burkholderia cepacia] WP_059923490.1 cytochrome C [Burkholderia stagnalis] (SEQ ID NO:10) WP_059565670.1 cytochrome C [Burkholderia stagnalis] OJD09200.1 cytochrome C [Burkholderia sp. DNA89] WP_060237338.1 cytochrome C [Burkholderia pseudomultivorans] WP_059707234.1 cytochrome C [Burkholderia cepacia] WP_060362538.1 cytochrome C [Burkholderia stagnalis] WP_060159353.1 cytochrome C [Burkholderia stagnalis] WP_069748432.1 cytochrome C [Burkholderia stabilis] WP_059677336.1 cytochrome C [Burkholderia cepacia] WP_059730424.1 cytochrome C [Burkholderia ubonensis] WP_059813883.1 cytochrome C [Burkholderia cepacia] WP_059993161.1 cytochrome C [Burkholderia stagnalis] WP_060236459.1 cytochrome C [Burkholderia ubonensis] WP_076476708.1 cytochrome C [Burkholderia ubonensis] WP_010089725.1 cytochrome C [Burkholderia ubonensis] WP_060195541.1 cytochrome C [Burkholderia ubonensis] WP_059971786.1 cytochrome C [Burkholderia pyrrocinia] WP_060367052.1 cytochrome C [Burkholderia ubonensis] WP_060016611.1 cytochrome C [Burkholderia ubonensis] WP_059924260.1 cytochrome C [Burkholderia ubonensis] WP_060048488.1 cytochrome C [Burkholderia ubonensis] WP_059892434.1 cytochrome C [Burkholderia ubonensis] WP_059488090.1 cytochrome C [Burkholderia ubonensis] WP_059483872.1 cytochrome C [Burkholderia ubonensis] WP_059865488.1 cytochrome C [Burkholderia ubonensis] WP_059633519.1 cytochrome C [Burkholderia ubonensis] WP_060287380.1 cytochrome C [Burkholderia ubonensis] WP_059853060.1 cytochrome C [Burkholderia ubonensis] WP_059968663.1 cytochrome C [Burkholderia ubonensis] WP_059752176.1 cytochrome C [Burkholderia ubonensis] WP_059532869.1 cytochrome C [Burkholderia ubonensis] WP_059660535.1 cytochrome C [Burkholderia ubonensis] WP_059590294.1 cytochrome C [Burkholderia ubonensis] WP_026043943.1 cytochrome C [Burkholderia pyrrocinia] WP_060264220.1 cytochrome C [Burkholderia ubonensis] WP_060019510.1 cytochrome C [Burkholderia ubonensis] WP_059946330.1 cytochrome C [Burkholderia ubonensis] WP_042588017.1 MULTISPECIES: cytochrome C [Burkholderia] WP_084908871.1 cytochrome C [[Pseudomonas] mesoacidophila] WP_071751088.1 cytochrome C [Burkholderia ubonensis] WP_060094261.1 cytochrome C [Burkholderia ubonensis] WP_059885808.1 cytochrome C [Burkholderia ubonensis] WP_059878406.1 cytochrome C [Burkholderia ubonensis] WP_071852856.1 cytochrome C [Burkholderia ubonensis] WP_060161154.1 cytochrome C [Burkholderia ubonensis] WP_059796027.1 cytochrome C [Burkholderia ubonensis] WP_059835010.1 cytochrome C [Burkholderia ubonensis] WP_059911385.1 cytochrome C [Burkholderia ubonensis] WP_059744627.1 cytochrome C [Burkholderia ubonensis] WP_045565069.1 cytochrome C [Burkholderia ubonensis] WP_060276465.1 cytochrome C [Burkholderia ubonensis] WP_060052180.1 cytochrome C [Burkholderia ubonensis] WP_059877204.1 cytochrome C [Burkholderia ubonensis] WP_059918860.1 cytochrome C [Burkholderia ubonensis] WP_059674217.1 cytochrome C [Burkholderia ubonensis] WP_059626644.1 cytochrome C [Burkholderia ubonensis] WP_059706954.1 cytochrome C [Burkholderia ubonensis] WP_059610359.1 cytochrome C [Burkholderia ubonensis] WP_071751781.1 cytochrome C [Burkholderia ubonensis] WP_060345076.1 cytochrome C [Burkholderia ubonensis] WP_060056668.1 cytochrome C [Burkholderia ubonensis] WP_059867004.1 cytochrome C [Burkholderia ubonensis] WP_059846198.1 cytochrome C [Burkholderia ubonensis] WP_059956839.1 cytochrome C [Burkholderia ubonensis] WP_059801215.1 cytochrome C [Burkholderia ubonensis] WP_060374144.1 cytochrome C [Burkholderia ubonensis] WP_060228155.1 cytochrome C [Burkholderia ubonensis] WP_060449283.1 cytochrome C [Burkholderia ubonensis] WP_060088315.1 cytochrome C [Burkholderia ubonensis] WP_060141594.1 cytochrome C [Burkholderia ubonensis] WP_059965520.1 cytochrome C [Burkholderia ubonensis] WP_059733305.1 cytochrome C [Burkholderia ubonensis] WP_059849627.1 cytochrome C [Burkholderia ubonensis] WP_060165437.1 cytochrome C [Burkholderia ubonensis] WP_059712923.1 cytochrome C [Burkholderia ubonensis] WP_059737856.1 cytochrome C [Burkholderia ubonensis] WP_059659999.1 cytochrome C [Burkholderia ubonensis] WP_059924960.1 cytochrome C [Burkholderia ubonensis] WP_059777863.1 cytochrome C [Burkholderia ubonensis] WP_060058770.1 cytochrome C [Burkholderia ubonensis] WP_059776014.1 cytochrome C [Burkholderia ubonensis] WP_060123897.1 cytochrome C [Burkholderia ubonensis] WP_060003887.1 cytochrome C [Burkholderia ubonensis] WP_071753220.1 cytochrome C [Burkholderia ubonensis] WP_069271183.1 cytochrome C [Burkholderia ubonensis] WP_060168536.1 cytochrome C [Burkholderia ubonensis] WP_059937950.1 cytochrome C [Burkholderia ubonensis] WP_059651010.1 cytochrome C [Burkholderia ubonensis] WP_059952547.1 cytochrome C [Burkholderia ubonensis] WP_059727989.1 cytochrome C [Burkholderia ubonensis] WP_060328321.1 cytochrome C [Burkholderia ubonensis] WP_059872364.1 cytochrome C [Burkholderia ubonensis] WP_059997503.1 cytochrome C [Burkholderia ubonensis] WP_071773630.1 cytochrome C [Burkholderia ubonensis] WP_059828379.1 cytochrome C [Burkholderia ubonensis] WP_060088886.1 cytochrome C [Burkholderia ubonensis] WP_060248705.1 cytochrome C [Burkholderia ubonensis] WP_036661765.1 cytochrome C [Pandoraea sp. SD6-2] WP_059573013.1 cytochrome C [Burkholderia sp. TSV86] WP_006027348.1 MULTISPECIES: cytochrome C [Burkholderia] WP_069235777.1 cytochrome C [Burkholderia sp. Bp7605] WP_059514380.1 cytochrome C [Burkholderia sp. TSV85] WP_063533919.1 cytochrome C [Burkholderia sp. MSMB1589WGS] WP_059929957.1 MULTISPECIES: cytochrome C [pseudomallei group] WP_060819951.1 cytochrome C [Burkholderia sp. BDU19] WP_059646659.1 MULTISPECIES: cytochrome C [pseudomallei group] WP_038746875.1 cytochrome C [Burkholderia sp. ABCPW 111] WP_059669953.1 cytochrome C [Burkholderia sp. MSMB1498] WP_038801472.1 cytochrome C [Burkholderia oklahomensis] WP_060356553.1 cytochrome C [Burkholderia sp. MSMB617WGS] WP_059582413.1 MULTISPECIES: cytochrome C [pseudomallei group] WP_085508044.1 cytochrome C [Burkholderia pseudomallei] WP_010118598.1 cytochrome C [Burkholderia oklahomensis] WP_059597682.1 cytochrome C [Burkholderia sp. BDU6] WP_025405690.1 cytochrome C [Burkholderia thailandensis] WP_038742304.1 cytochrome C [Burkholderia pseudomallei] WP_009900297.1 cytochrome C [Burkholderia thailandensis] (SEQ ID NO:11) WP_004532924.1 cytochrome C [Burkholderia pseudomallei] WP_058034623.1 cytochrome C [Burkholderia pseudomallei] WP_038758421.1 cytochrome C [Burkholderia pseudomallei] WP_038760788.1 cytochrome C [Burkholderia pseudomallei] WP_004540659.1 cytochrome C [Burkholderia pseudomallei] WP_085547264.1 cytochrome C [Burkholderia pseudomallei] WP_076883397.1 cytochrome C [Burkholderia pseudomallei] WP_059473995.1 cytochrome C [Burkholderia sp. BDU5] WP_041191039.1 cytochrome C [Burkholderia pseudomallei] WP_043296725.1 cytochrome C [Burkholderia thailandensis] WP_063597172.1 cytochrome C [Burkholderia pseudomallei] WP_066496408.1 cytochrome C [Burkholderia sp. BDU8] WP_038799895.1 cytochrome C [Burkholderia pseudomallei] WP_004539114.1 cytochrome C [Burkholderia pseudomallei] WP_004524074.1 cytochrome C [Burkholderia pseudomallei] WP_017881863.1 cytochrome C [Burkholderia pseudomallei] WP_009897184.1 cytochrome C [Burkholderia thailandensis] WP_004536717.1 cytochrome C [Burkholderia pseudomallei] WP_076885712.1 cytochrome C [Burkholderia pseudomallei] WP_066570748.1 cytochrome C [Burkholderia sp. ABCPW 14] WP_076903220.1 cytochrome C [Burkholderia pseudomallei] WP_076936464.1 cytochrome C [Burkholderia pseudomallei] WP_076893617.1 cytochrome C [Burkholderia pseudomallei] WP_043299328.1 cytochrome C [Burkholderia pseudomallei] WP_038785043.1 cytochrome C [Burkholderia pseudomallei] WP_041220968.1 cytochrome C [Burkholderia pseudomallei] WP_076891198.1 cytochrome C [Burkholderia pseudomallei] WP_076883909.1 cytochrome C [Burkholderia pseudomallei] WP_004528232.1 cytochrome C [Burkholderia pseudomallei] WP_085539130.1 cytochrome C [Burkholderia pseudomallei] WP_038784065.1 cytochrome C [Burkholderia pseudomallei] WP_038760183.1 cytochrome C [Burkholderia pseudomallei] WP_038730591.1 cytochrome C [Burkholderia pseudomallei] WP_038765827.1 cytochrome C [Burkholderia pseudomallei] WP_076855330.1 cytochrome C [Burkholderia pseudomallei] WP_004199672.1 cytochrome C [Burkholderia mallei] KGC89207.1 cytochrome C family protein [Burkholderia pseudomallei] ED093432.1 glucose dehydrogenase, beta subunit [Burkholderia pseudomallei Pasteur 52237] WP_011205494.1 cytochrome C subunit II [Burkholderia pseudomallei] KGV82938.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4375] CDU31054.1 putative cytochrome C subunit II [Burkholderia pseudomallei] KGV67566.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4299] EDK84065.1 cytochrome C family protein [Burkholderia mallei 2002721280] WP_074287454.1 cytochrome C [Burkholderia sp. GAS332] WP_035557403.1 cytochrome C [Burkholderia sp. 9120] WP_084534186.1 cytochrome C [Paraburkholderia dilworthii] WP_017774215.1 cytochrome C [Paraburkholderia kururiensis] WP_030100524.1 cytochrome C [Burkholderia sp. K24] WP_028194542.1 cytochrome C [Paraburkholderia fungorum] WP_046573324.1 cytochrome C [Paraburkholderia fungorum] WP_042300635.1 cytochrome C [Paraburkholderia kururiensis] WP_084166897.1 cytochrome C [Paraburkholderia caledonica] SDI05126.1 cytochrome C, mono- and diheme variants [Paraburkholderia phenazinium] WP_051120977.1 cytochrome C [Paraburkholderia bryophila] WP_073428494.1 MULTISPECIES: cytochrome C [Burkholderiaceae] WP_074300113.1 cytochrome C [Paraburkholderia phenazinium] WP_074768708.1 cytochrome C [Paraburkholderia fungorum] WP_075465130.1 cytochrome C [Ralstonia solanacearum] WP_039597687.1 cytochrome C [Ralstonia sp. A12] WP_055334967.1 cytochrome C [Ralstonia solanacearum] WP_078223437.1 cytochrome C [blood disease bacterium A2-HR MARDI] WP_013213209.1 cytochrome C [Ralstonia solanacearum] WP_063393008.1 cytochrome C [Ralstonia mannitolilytica] WP_003265144.1 cytochrome C, partial [Ralstonia solanacearum] (SEQ ID NO:12) WP_039568931.1 cytochrome C [Ralstonia solanacearum] WP_045786290.1 cytochrome C [Ralstonia mannitolilytica] WP_064802148.1 cytochrome C [Ralstonia insidiosa] WP_021195198.1 MULTISPECIES: cytochrome C [Ralstonia] WP_004629446.1 cytochrome C, mono- and diheme variants family [Ralstonia pickettii] WP_012435095.1 cytochrome C [Ralstonia pickettii] WP_003279246.1 cytochrome C [Ralstonia solanacearum] WP_048931829.1 cytochrome C [Ralstonia sp. MD27] WP_027677928.1 cytochrome C [Ralstonia sp. UNC404CL21Col] WP_012761509.1 cytochrome C [Ralstonia pickettii] (SEQ ID NO:13) WP_045204557.1 cytochrome C [Burkholderiaceae bacterium 26] CUV46938.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum] WP_024973348.1 cytochrome C [Ralstonia pickettii] AKZ27209.1 cytochrome C [Ralstonia solanacearum] WP_020749403.1 oxidoreductase dehydrogenase (cytochrome C subunit) [Ralstonia solanacearum] WP_024976325.1 cytochrome C [Ralstonia pickettii] WP_049842155.1 cytochrome C [Ralstonia solanacearum] WP_028852719.1 cytochrome C [Ralstonia solanacearum] WP_071895582.1 cytochrome C [Ralstonia solanacearum] SFP41323.1 cytochrome C, mono- and diheme variants [Ralstonia sp. NFACC01] WP_064477581.1 cytochrome C [Ralstonia solanacearum] WP_058908222.1 cytochrome C [Ralstonia solanacearum] WP_009238766.1 MULTISPECIES: cytochrome C [Ralstonia] CUV21878.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum] WP_011000726.1 cytochrome C [Ralstonia solanacearum] WP_071507651.1 cytochrome C [Ralstonia solanacearum] WP_065857157.1 cytochrome C [Ralstonia pickettii] WP_019717689.1 cytochrome C [Ralstonia solanacearum] WP_071012822.1 cytochrome C [Ralstonia solanacearum] WP_020831436.1 2-Keto-D-gluconate dehydrogenase [Ralstonia solanacearum] CUV55668.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]

Examples of the mutant cytochrome c protein also include a protein obtained by modifying an amino acid sequence having a sequence identity of not less than 60% to SEQ ID NO:4 such as one described above, for example, the amino acid sequence of any of SEQ ID NO:6 to 13, such that the protein lacks the first and second heme-binding domains as counted from the amino-terminal side. For example, the mutant cytochrome c protein may be the same as any of the amino acid sequences of SEQ ID NOs:6 to 13 except that the protein lacks the first and second heme-binding domains, or may be the same as any of the amino acid sequences of SEQ ID NOs:6 to 13 except that the protein lacks the region comprising the first and second heme-binding domains (the region corresponding to amino acids 43 to 195 of SEQ ID NO:4).

The original sequence to be modified may be the amino acid sequence of each of SEQ ID NOs:6 to 13 having a substitution(s), deletion(s), addition(s), and/or insertion(s) of one or several amino acids in a region(s) other than the modification site (i.e. the site of modification such that the protein lacks the first and second heme-binding domains) and the third heme-binding domain.

Thus, as long as the electron transfer function is maintained, the mutant cytochrome c protein may be a protein having the same amino acid sequence as any of the amino acid sequences of SEQ ID NOs:6 to 13 except that the protein lacks the first and second heme-binding domains and that one or several amino acids are substituted, deleted, added, and/or inserted at a position(s) in a region(s) other than the third heme-binding domain, or may be a protein having the same amino acid sequence as any of the amino acid sequences of SEQ ID NOs:6 to 13 except that the protein lacks the region containing the first and second heme-binding domains (the region corresponding to amino acids 43 to 195 of SEQ ID NO:4) and that one or several amino acids are substituted, deleted, added, and/or inserted at a position(s) in a region(s) other than the third heme-binding domain. The term “one or several” herein means, for example, 1 to 20, 1 to 10, 1 to 8, 1 to 5, or 1 to 3, i.e. 1, 2 or 3. The substitution may be conservative substitution. “Conservative substitution” means substitution between amino acids having similar properties, such as substitution between acidic amino acids, substitution between neutral amino acids, or substitution between basic amino acids.

<Oxidoreductase-Cytochrome Complex>

The mutant cytochrome protein according to one embodiment of the present invention may be used together with a catalytic subunit of an oxidoreductase, and is capable of receiving an electron generated by oxidation-reduction reaction and then transferring the electron to an electrode. Thus, an oxidoreductase-cytochrome complex containing the mutant cytochrome protein can be used as an electrochemical sensor. The electrochemical sensor may be a glucose sensor containing the mutant cytochrome protein and glucose oxidoreductase.

<Oxidoreductase>

The oxidoreductase is not limited as long as it is an enzyme capable of oxidation-reduction of a measurement target substance. Examples of its catalytic subunit include catalytic subunits of enzymes containing at least one of pyrrolo-quinoline quinone (PQQ) and flavin adenine dinucleotide (FAD). Examples of an oxidoreductase containing PQQ include PQQ glucose dehydrogenase (PQQGDH), and examples of an oxidoreductase containing FAD include cytochrome glucose dehydrogenase (CyGDH) having an α-subunit containing FAD and glucose oxidase (GOD). Other examples of the oxidoreductase include cholesterol oxidase, quinoheme ethanol dehydrogenase (QHEDH, PQQ Ethanol dh), sorbitol dehydrogenase (Sorbitol DH), D-fructose dehydrogenase (Fructose DH), cellobiose dehydrogenase, and lactate dehydrogenase.

Among these, glucose dehydrogenase (GDH) is preferred.

The glucose dehydrogenase is not limited as long as it has the glucose dehydrogenase activity. Glucose dehydrogenase catalytic subunits derived from various organisms, including those already known, may be used. It may be derived from the same microorganism as the microorganism from which the mutant cytochrome protein is derived. More specifically, for example, an α-subunit protein derived from the Burkholderia cepacia KS1 strain having the amino acid sequence of SEQ ID NO:3 may be used. However, as long as the subunit can function as an α-subunit of GDH, it may be a protein having the same amino acid sequence as the amino acid sequence of SEQ ID NO:3 except that one or several amino acid residues are substituted, deleted, inserted, and/or added. Further, as long as the subunit can function as an α-subunit of GDH, it may be a protein having the same amino acid sequence as the amino acid sequence of an α-subunit of a strain other than the KS1 strain except that one or several amino acid residues are substituted, deleted, inserted, and/or added. The term “one or several” means, for example, 1 to 20, 1 to 10, or 1 to 5, i.e. 1, 2, 3, 4 or 5. A number of mutations are known to increase the enzymatic activity or the substrate specificity of the α-subunit protein of glucose dehydrogenase. α-Subunit proteins of glucose dehydrogenase having such mutations may be used.

The glucose dehydrogenase complex may further contain a γ-subunit in addition to the mutant cytochrome protein and the catalytic subunit. The γ-subunit is not limited as long as it functions as a γ-subunit, and γ-subunits derived from various organisms, including those already known, may be used. It may be derived from the same microorganism as the microorganism from which the mutant cytochrome protein is derived. More specifically, for example, a protein derived from the Burkholderia cepacia KS1 strain having the amino acid sequence of SEQ ID NO:2 may be used. However, as long as the subunit can function as a γ-subunit, it may be a protein having the same amino acid sequence as the amino acid sequence of SEQ ID NO:2 except that one or several amino acid residues are substituted, deleted, inserted, and/or added. Further, as long as the subunit can function as a γ-subunit, it may be a protein having the same amino acid sequence as the amino acid sequence of a γ-subunit of a strain other than the KS1 strain except that one or several amino acid residues are substituted, deleted, inserted, and/or added. The term “one or several” means, for example, 1 to 15, 1 to 10, or 1 to 5, i.e. 1, 2, 3, 4 or 5. Alternatively described, as long as the subunit can function as a γ-subunit, it may be a protein having an amino acid identity of not less than 60%, preferably not less than 80%, more preferably not less than 90% or 95% to SEQ ID NO:2. The term “functions as a γ-subunit” means that, when a complex is formed together with an α-subunit(s), the γ-subunit has a function to increase the GDH activity of the complex.

<DNA>

The present invention also provides a DNA encoding the mutant cytochrome protein. The nucleotide sequence of the DNA encoding the mutant cytochrome protein can be specified based on the amino acid sequence of the mutant cytochrome protein. For example, the DNA can be obtained by modifying the nucleotide sequence of a DNA encoding a wild-type cytochrome protein such that the protein lacks the first and second heme-binding domains or a region(s) containing these.

Specific examples of the DNA encoding a cytochrome protein of Burkholderia cepacia include a DNA containing the nucleotide sequence of nucleotides 2386 to 3660 of SEQ ID NO:1, which encodes a β-subunit of glucose dehydrogenase. The DNA encoding a β-subunit is not limited to the DNA having the nucleotide sequence of nucleotides 2386 to 3660 of SEQ ID NO:1, and may be a DNA which hybridizes, under stringent conditions, with the DNA having the complementary sequence of this nucleotide sequence, and which encodes a protein that can function as a β-subunit.

Examples of the DNA encoding the mutant cytochrome protein include the DNA consisting of the nucleotide sequence of nucleotides 3372 to 3660 or 3325 to 3660 of SEQ ID NO:5, and a DNA which hybridizes, under stringent conditions, with the DNA having the complementary sequence of this nucleotide sequence, and which encodes a protein that can function as the mutant cytochrome protein.

A mutant cytochrome protein having a desired mutation can be obtained by constructing a mutant DNA by, for example, introduction of a desired amino acid deletion(s) to a DNA encoding a cytochrome protein by site-directed mutagenesis, or amplification of only a particular region of the DNA by PCR, and then allowing expression from the mutant DNA using an appropriate expression system.

In cases where an oxidoreductase-cytochrome complex containing the mutant cytochrome protein is used, it is preferred to use a DNA encoding the catalytic subunit of the oxidoreductase together with a DNA encoding the mutant cytochrome protein.

The DNA encoding the catalytic subunit of the oxidoreductase is not limited, and may be appropriately selected and used depending on the purpose. Examples of the DNA include a DNA encoding a glucose dehydrogenase α-subunit having the nucleotide sequence of nucleotides 764 to 2380 of SEQ ID NO:1. The α-subunit gene may be a DNA which hybridizes, under stringent conditions, with the DNA having the complementary sequence of the nucleotide sequence of nucleotides 764 to 2380 of the nucleotide sequence of SEQ ID NO:1, and which encodes a protein having the GDH activity.

In cases where a glucose dehydrogenase γ-subunit is contained in the oxidoreductase-cytochrome complex, it is preferred to use a DNA encoding the γ-subunit of the oxidoreductase together with a DNA encoding the mutant cytochrome protein and a DNA encoding the catalytic subunit.

Specific examples of the DNA encoding the γ-subunit include a DNA containing the nucleotide sequence of nucleotides 258 to 761 of SEQ ID NO:1. The DNA encoding the γ-subunit may be a DNA which hybridizes, under stringent conditions, with the DNA having the complementary sequence of the nucleotide sequence of nucleotides 258 to 761 of SEQ ID NO:1, and which encodes a protein that can function as a γ-subunit.

Examples of the stringent conditions described above include conditions that allow hybridization of DNAs having an identity of not less than 80%, not less than 90%, or not less than 95%, with each other. Specific examples of the stringent conditions include those in which hybridization reaction is followed by washing with 0.1×SSC and 0.1% SDS at 60° C. Alternatively, preferred DNA has the above described sequence identity to the parent DNA, i.e. to the DNA to which hybridization was contemplated.

The DNA encoding the cytochrome protein, the DNA encoding the catalytic subunit of oxidoreductase, and the like can be obtained by, for example, using chromosomal DNA of a microorganism such as Burkholderia cepacia as a template for PCR or for the hybridization method.

The nucleotide sequence of a chromosomal DNA fragment containing the GDH γ-subunit gene, α-subunit gene, and β-subunit gene of the Burkholderia cepacia KS1 strain is shown in SEQ ID NO:1. This nucleotide sequence has three open reading frames (ORFs). As counted from the 5′-end side, the first ORF encodes the γ-subunit (SEQ ID NO:2); the second ORF encodes the α-subunit (SEQ ID NO:3); and the third ORF encodes the β-subunit (SEQ ID NO:4).

The mutant cytochrome protein and the catalytic subunit may be separately expressed from their DNAs, or may be expressed using a DNA that polycistronically contains the DNA encoding the mutant cytochrome protein and the DNA encoding the catalytic subunit.

<Vector>

The vector used for obtaining the genes of the mutant cytochrome protein and the catalytic subunit, introducing the mutations, expressing the genes, and the like is not limited as long as the vector can function in the host microorganism. Examples of the vector include those that can function in bacteria belonging to the genus Escherichia, such as pTrc99A, pBR322, pUC18, pUC118, pUC19, pUC119, pACYC184, and pBBR122. The promoter to be used for the gene expression may also be appropriately selected depending on the host. Examples of the promoter include lac, trp, tac, trc, PL, tet, and PhoA.

<Transformant>

By introducing the DNA described above or a vector containing it into a host microorganism to obtain a transformant, the mutant cytochrome protein or the oxidoreductase-cytochrome complex can be expressed. For the transformation, a known method may be employed. Examples of the method include the competent cell method by calcium treatment, the protoplast method, and the electroporation method.

The host microorganism is not limited as long as it can be used for protein expression. Examples of the host microorganism include, but are not limited to, bacteria belonging to the genus Escherichia such as Escherichia coli; bacteria belonging to the genus Bacillus such as Bacillus subtilis; yeasts such as Saccharomyces cerevisiae and Pichia pastoris; and filamentous fungi such as Aspergillus niger and Aspergillus oryzae.

<Biosensor>

The mutant cytochrome protein according to one embodiment of the present invention can be used for an electrochemical biosensor that utilizes oxidation-reduction reaction. Regarding the type of the biosensor, the biosensor may be used for a variety of uses depending on the type of the oxidoreductase used in combination. For example, in cases where glucose oxidoreductase is used as the oxidoreductase, a glucose sensor may be provided. In cases where lactate dehydrogenase is used as the oxidoreductase, a lactate sensor may be provided.

Among these, a glucose sensor is preferred. In cases where a glucose sensor is prepared, the mutant cytochrome protein and a catalytic subunit of the oxidoreductase may be contained in the reagent layer of the enzyme electrode. Further, a γ-subunit may be contained. By this, glucose in the sample can be oxidized by the catalytic subunit. The electron generated thereby is accepted by the mutant cytochrome protein and then transferred to the electrode. As a result, a response current depending on the glucose concentration in the sample flows, and the glucose concentration can then be calculated based on the current value.

Specific examples of the glucose sensor include glucose sensors that use, as a working electrode, an enzyme electrode formed by immobilization of a complex containing the mutant cytochrome protein and a catalytic subunit of glucose dehydrogenase on a surface of an electrode such as a gold electrode, platinum electrode, or carbon electrode. The sensor means a measurement system for electrochemically measuring the concentration of a test substance of interest, and may be a system containing the following three electrodes: a working electrode (enzyme electrode), a counter electrode (platinum or the like), and a reference electrode (Ag/AgCl or the like). The sensor may also be a two-electrode system constituted by a working electrode and a counter electrode, such as those used in conventional, simple blood glucose level systems. The sensor may further contain a constant-temperature cell in which a buffer and a test sample are to be placed; a power source for applying an electric potential to the working electrode; an ammeter; a recorder; and/or the like. The sensor may be either a batch-type sensor or a flow-type sensor. The flow-type sensor may be a sensor which can continuously measure the blood glucose level. That is, the sensor may be a sensor having a two-electrode system or a three-electrode system on which the enzyme containing the mutant cytochrome protein is immobilized, which electrode system is inserted into a blood sample or a dialysis sample that is continuously supplied, or into blood or interstitial fluid, to perform the measurement. The structure of such an enzyme sensor is well known in the art, and described in, for example, Biosensors—Fundamental and Applications—Anthony P. F. Turner, Isao Karube and Geroge S. Wilson, Oxford University Press 1987.

The measurement of the glucose concentration using the glucose sensor according to one embodiment of the present invention can be carried out, for example, as follows. A buffer is placed in a constant-temperature cell of the sensor, and the temperature of the cell is kept constant. As a working electrode, an enzyme electrode on which the mutant cytochrome protein and a catalytic subunit protein of glucose oxidoreductase are immobilized is used. As a counter electrode, for example, a platinum electrode is used. As a reference electrode, for example, an Ag/AgCl electrode is used. A constant electric potential is applied to the working electrode (for example, by application of 0 to +300 mV, 0 to +150 mV, or 0 to +100 mV with respect to a silver-silver chloride electrode). After the electric current becomes constant, a sample containing glucose is placed in the constant-temperature cell, and the increase in the electric current is measured. According to a calibration curve prepared using glucose solutions having standard concentrations, the glucose concentration in the sample can be calculated.

A GDH complex containing the mutant cytochrome protein and a GDH catalytic subunit can also be used as a component of a glucose assay kit. The glucose assay kit may contain a coloring or luminescence reagent, a dilution buffer, a standard substance, manufacturer's instructions, and/or the like in addition to the GDH complex containing the mutant cytochrome protein and a GDH catalytic subunit.

For example, a glucose sensor and a glucose assay kit using the wild-type GDH of Burkholderia cepacia are described in US 2004/0023330 A1. The GDH containing the mutant cytochrome protein can be similarly used.

EXAMPLES

More concrete embodiments of the present invention are described below by referring to Examples. However, embodiments of the present invention are not limited to these Examples.

[Example 1] Construction of Mutant GDH β-Subunit Gene

As a plasmid to be used for construction of a mutant GDH β-subunit gene, pTrc99Aγα(QYY)β, which was prepared by introducing mutations to the α-subunit in the plasmid pTrc99Aγαβ described in JP 2012-090563 A, was used. This plasmid is a plasmid prepared by inserting a DNA fragment containing in frame the GDH γ-subunit structural gene, the α-subunit structural gene, and the β-subunit structural gene isolated from chromosomal DNA of the Burkholderia cepacia KS1 strain (FERM BP-7306) into the cloning site of the pTrc99A vector, wherein in the α-subunit structural gene, the codons encoding the serine at the 326th residue, the serine at the 365th residue, and the alanine at the 472nd residue are substituted with codons encoding glutamine, tyrosine, and tyrosine, respectively. The structural genes of GDH in this plasmid are regulated by the trc promoter. pTrc99Aγαβ has an ampicillin resistance gene.

First, using the plasmid DNA described above as a template, and the oligonucleotides having the following sequences as primers, PCR was carried out to amplify a DNA fragment encoding the γ-subunit, the α-subunit, and the signal sequence of the β-subunit of GDH.

[Forward primer] (SEQ ID NO: 14) 5′-ACGTAGCCATGGCACACAACGACAACCACTC-3′ [Reverse primer] (SEQ ID NO: 15) 5′-ATCGGCCGCGCGCGCGAAGCCCGGCAA-3′

The PCR was carried out using the reaction composition shown below by incubation at 95° C. for 30 seconds, 25 cycles of incubation at 95° C. for 30 seconds, at 55° C. for 1 minute, and then at 72° C. for 2 minutes, and then incubation at 72° C. for 5 minutes, followed by keeping the reaction liquid at 4° C.

[Reaction Liquid Composition]

pTrc99Aγα(QYY)β (100 ng/μl) 0.5 μl 10 × Reaction buffer 2 μl Forward primer (100 ng/μl) 0.5 μl Reverse primer (100 ng/μl) 0.5 μl dNTP 0.4 μl Distilled water 15.7 μl DNA polymerase 0.4 μl Total 20 μl

Further, DNA fragments encoding the third heme-binding domain of the β-subunit, that is, the region of amino acids 314 to 425 or 330 to 425 of SEQ ID NO:4, were amplified by PCR using the oligonucleotides having the sequences shown below as primers. Each of the forward primers has a sequence corresponding to the signal sequence of the β-subunit at its 5′-end side.

[Forward primer for amplification of the region from 314 to 425] (SEQ ID NO: 16) 5′-TTGCCGGGCTTCGCGCGCGCGGCCGATCTGCGCGGTGTCGCGCTCG CG-3′ [Forward primer for amplification of the region from 330 to 425] (SEQ ID NO: 17) 5′-TTGCCGGGCTTCGCGCGCGCGGCCGATTATCTCGGCAACTGCGCGA CG-3′ [Reverse Primer] (SEQ ID NO: 18) 5′-GTGGTGCTCGAGTGCGGCCGCGCGCAGCTTCGCGACGTCCTG-3′

The PCR was carried out using the reaction composition shown below by incubation at 95° C. for 30 seconds, 25 cycles of incubation at 95° C. for 30 seconds, at 55° C. for 1 minute, and then at 72° C. for 30 seconds, and then incubation at 72° C. for 5 minutes, followed by keeping the reaction liquid at 4° C.

[Reaction Liquid Composition]

pTrc99Aγα(QYY)β (100 ng/μl) 0.5 μl 10 × Reaction buffer 2 μl Forward primer (100 ng/μl) 0.5 μl Reverse primer (100 ng/μl) 0.5 μl dNTP 0.4 μl Distilled water 15.7 μl DNA polymerase 0.4 μl Total 20 μl

The DNA fragments obtained by the above operations were separately purified and then mixed together to provide a template, which was used to perform overlap extention PCR using the oligonucleotides having the sequences shown below as primers, thereby linking and amplifying the DNA fragments. The reverse primer was designed to have a histidine tag sequence such that a histidine tag is added to the C-terminus of the mutant β-subunit.

[Forward primer] (SEQ ID NO: 14) 5′-ACGTAGCCATGGCACACAACGACAACCACTC-3′ [Reverse primer] (SEQ ID NO: 19) 5′-GTACGTAAGCTTTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCC GC-3′

The PCR was carried out using the reaction composition shown below by incubation at 95° C. for 30 seconds, 25 cycles of incubation at 95° C. for 30 seconds, at 55° C. for 1 minute, and then at 72° C. for 4 minutes, and then incubation at 72° C. for 5 minutes, followed by keeping the reaction liquid at 4° C.

[Reaction Liquid Composition]

PCR product of the region from the γ-subunit to 1 μl the β-subunit signal sequence (100 ng/μl) PCR product of the β-subunit 314 to 425 or PCR 1 μl product of the-β subunit 330 to 425 (100 ng/μl) 10 × Reaction buffer 2 μl Forward primer (100 ng/μl) 0.5 μl Reverse primer (100 ng/μl) 0.5 μl dNTP 0.4 μl Distilled water 14.2 μl DNA polymerase 0.4 μl Total 20 μl

After purification of the PCR product obtained, the N-terminal side was digested with NcoI, and the C-terminal side was digested with HindIII, followed by ligating the digestion product into pTrc99A which had been treated with the same enzymes. Escherichia coli DH5a was transformed with the obtained recombinant vector. Colonies formed on LB agar medium supplemented with 50 μg/mL carbenicillin were collected. The transformants obtained were cultured in liquid LB medium, and plasmids were extracted therefrom. Thereafter, the DNA fragments inserted therein were analyzed. As a result, insertion of the sequence covering γ-subunit, the α-subunit, and the signal sequence of the β-subunit, as well as the gene encoding the 314th to 425th residues of the β-subunit or the gene encoding the 330th to 425th residues of the β-subunit, was confirmed. These plasmids were designated pTrc99Aγα(QYY)β314-His and pTrc99Aγα(QYY)β330-His, respectively. The structural genes of GDH in these plasmids have a histidine tag at the C-terminus of the β-subunit, and are regulated by the trc promoter. Each plasmid has an ampicillin resistance gene.

[Example 2] Expression of Burkholderia cepacia GDH Containing Mutant GDH β-Subunit

A GDH containing a mutant GDH β-subunit gene was produced using the expression plasmid obtained in Example 1.

The Escherichia coli BL21 (DE3) strain to which the GDH expression plasmid containing the mutant GDH β-subunit gene and the plasmid pBBJMccm having genes indispensable for maturation of cytochrome c were introduced was cultured in 3 ml of LB medium (supplemented with 50 μg/ml carbenicillin and 50 μg/ml kanamycin) with shaking at 37° C. overnight using a test tube. The resulting culture liquid was inoculated at 1% to two 300-ml Sakaguchi flasks each containing 50 ml of a medium prepared such that the composition per 1 L of the culture liquid was as shown in Table 1 (supplemented with 50 μg/ml carbenicillin and 50 μg/ml kanamycin), and shake culture was carried out at 25° C. for 28 hours.

However, in cases where the host can constantly allow maturation of cytochrome c, for example, in cases where the transformant has the ccm gene inserted into the genome by homologous recombination or the like and is constantly expressing it, vectors such as pBBJMccm are not necessary.

TABLE 1 Medium Composition (per 1 L) Tryptone 10 g Yeast extract 5 g Sodium chloride 5 g Glycerin 0.5 g Glucose 0.05 g Lactose monohydrate 0.21 g Ammonium sulfate 0.33 g Potassium dihydrogen phosphate 0.68 g Disodium hydrogen phosphate dodecahydrate 1.79 g Magnesium sulfate 0.025 g

The bacterial cells were collected from the culture liquid obtained by the culture described above, and BugBuster Protein Extraction reagent (Merck Millipore) was added thereto at 5 ml per 1 g of the wet bacterial cells obtained, followed by suspending and lysing the bacterial cells. The resulting suspension was centrifuged (15,000 rpm, 20 minutes, 4° C.), and then the residue was discarded to provide a crude enzyme sample.

Regarding γα(QYY)β314-His, the crude enzyme sample was dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.0) supplemented with 0.5 M sodium chloride and 20 mM imidazole, and then added to a column packed with Ni-NTA agarose (QIAGEN) equilibrated with the same buffer. After washing with 20 mM sodium phosphate buffer (pH 7.0) supplemented with 0.5 M sodium chloride and 54 mM imidazole, the sample was eluted with 20 mM sodium phosphate buffer (pH 7.0) supplemented with 0.5 M sodium chloride and 140 mM imidazole, to obtain a purified enzyme sample.

[Example 3] Functional Analysis of Burkholderia cepacia GDH Containing Mutant GDH β-Subunit

Measurement of the GDH activity was carried out by the PMS/DCIP system and the ruthenium (Ru)/MTT system. The former system shows the general GDH activity, and the latter system shows the GDH activity based on the electron transfer to a mediator through the β-subunit.

The activity measurement in the PMS/DCIP system was carried out as follows. To 20 mM potassium phosphate buffer (pH 7.0), an enzyme sample, methylphenazine methosulfate (PMS) (final concentration, 0.6 mM), 2,6-dichlorophenol indophenol (DCIP) (final concentration, 0.06 mM), and glucose (final concentration, 4 mM or 40 mM) were added, and the degree of change in the absorbance at 600 nm, which is the absorption wavelength derived from DCIP, per 1 minute was measured using a spectrophotometer.

The activity measurement in the Ru/MTT system was carried out as follows. To 20 mM potassium phosphate buffer (pH 7.0), an enzyme sample, hexaammineruthenium (III) chloride (final concentration, 2%), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (final concentration, 1 mM), and glucose (final concentration, 4 mM or 40 mM) were added, and the amount of change in the absorbance at 565 nm, which is the absorption wavelength derived from formazan produced from MTT, per 1 minute was measured using a spectrophotometer.

The results of activity measurement of the crude enzyme samples are shown in Table 2.

TABLE 2 Enzyme activity (U/mg) at each substrate concentration PMS/DCIP Ru/MTT 4 mM 40 mM 4 mM 40 mM mg/ml Glc Glc Glc Glc γα(QYY)β-His 7.00 5.35 19.28 1.21 3.24 γα(QYY)β314-His 5.08 5.40 12.01 0.14 0.20 γα(QYY)β330-His 4.03 4.18 10.0 0.11 0.11

As shown in Table 2, both mutants exhibited the GDH activity in the Ru/MTT system although the activity was lower than that of the enzyme containing the wild-type β-subunit. Thus, it was suggested that these mutants are capable of electron transfer to the mediator through the β-subunit. In wild-type enzyme, a γα complex containing no β-subunit showed an enzyme activity of about 45 U/mg when PMS/DCIP was used as an electron acceptor (Biochimica et Biophysica Acta, 1645(2), 133-138.). This corresponded to only about 15% of enzyme activity as compared to the value observed for the complex containing the β-subunit (about 300 U/mg; Journal of Biotechnology, 123(2), 127-136.). In contrast, each complex containing a mutant cytochrome c protein showed almost the same activity as that observed for the complex containing the wild-type β-subunit. It was thus found that the mutant β-subunit receives an electron from the α-subunit, and then transfers the electron to PMS/DCIP.

γα(QYY)β314 was purified, and its enzyme activity for 40 mM glucose was measured according to the procedure described above. The measurement was also carried out for γα(QYY)β separately prepared, which contains the wild-type β-subunit. The results are shown in Table 3.

TABLE 3 Enzyme activity (U/mg) mg/ml PMS/DCIP Ru/MTT γαQYYβ-His 7.79 644.3 55.0 γα(QYY)β314-His 8.69 154.0 18.7

The purified γα(QYY)β314 showed the GDH activity in the Ru/MTT system, and its ratio to the GDH activity in the PMS/DCIP system was similar to that in the case of the enzyme complex containing the wild-type β-subunit. It was thus suggested that this mutant allows electron transfer to the mediator through the truncated β-subunit similarly to the enzyme complex containing the wild-type β-subunit.

[Example 4] Preparation of Glucose Sensor

An enzyme electrode in which GDH containing a mutant β-subunit is immobilized on a gold surface through a monolayer-forming molecule was prepared. As the monolayer-forming molecule, DSH, which is as shown below, was used.

More specifically, a gold wire (diameter, 0.5 mm; length, 6 to 7 cm) was immersed in Piranha solution (200 μl) at room temperature for 2 hours, and then washed with acetone. The gold wire was then immersed in a solution of DSH in acetone (concentration, 20 μM), and incubated at 25° C. for 24 hours to allow binding of thiol groups of DSH to the gold surface. Subsequently, the gold wire was washed with acetone, and then immersed in phosphate buffer (300 μl) containing the GDH α,β-subunit complex (concentration, 0.03 mg/ml), followed by incubation at 4° C. overnight to allow binding of the enzyme complex through a functional group of DSH, thereby obtaining an enzyme electrode.

[Example 5] Measurement of Glucose Concentration

Using the enzyme electrode described above, the response current value for 0 mM (background), 1 mM, 5 mM, or 50 mM aqueous glucose solution was measured by chronoamperometry. The glucose measurement was carried out at 37° C. using a counter electrode (Pt wire) and a reference electrode (silver/silver chloride) by application of an electric potential of 0 V, +0.1 V, or +0.4 V (vs. silver/silver chloride) to the working electrode.

<Results>

Using as a reference the glucose concentration properties of the current value obtained by the application of 400 mV with respect to the silver-silver chloride electrode, the current values at 0 mV and 100 mV were compared.

As a result, as shown in FIG. 1, in the case of application of an electric potential of 0 mV, the Comparative Example (dotted line, glucose sensor containing a glucose dehydrogenase (GDH) complex having a wild-type β-subunit) did not show an oxidation current dependent on the glucose concentration, but the Example (solid line) showed an oxidation current dependent on the glucose concentration.

In the case of application of an electric potential of 100 mV, the Comparative Example also showed an oxidation current dependent on the glucose concentration, but the Example showed a higher electric current value output.

It was thus shown that the sensor using the truncated β-subunit in the Example enables acquisition of the catalytic electric current even at a lower oxidation potential compared to the sensor using the wild-type β-subunit.

INDUSTRIAL APPLICABILITY

The biosensor using the mutant cytochrome protein according to one embodiment of the present invention is capable of measuring the electric current at a low electric potential, and may be used for biosensors such as glucose sensors.

While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to one skilled in the art that various changes may be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents as well as JP2017-131345 is incorporated by reference herein in its entirety. 

What is claimed is:
 1. A mutant cytochrome protein originated from a cytochrome protein having three heme-binding domains, the mutant cytochrome protein lacking the first heme-binding domain and the second heme-binding domain as counted from the N-terminus.
 2. The mutant cytochrome protein according to claim 1, wherein said mutant cytochrome protein lacks a region(s) comprising the first and second heme-binding domains.
 3. The mutant cytochrome protein according to claim 2, wherein the region comprising the first and second heme-binding domains corresponds to the region of amino acids from 43 to 195 of SEQ ID NO:4.
 4. The mutant cytochrome protein according to claim 1, wherein the cytochrome protein having three heme-binding domains is a cytochrome c protein.
 5. The mutant cytochrome protein according to claim 4, wherein the cytochrome c protein is that of a Burkholderia microorganism.
 6. The mutant cytochrome protein according to claim 5, wherein the cytochrome c protein is that of Burkholderia cepacia.
 7. The mutant cytochrome protein according to claim 1, wherein the original cytochrome protein having three heme-binding domains comprises an amino acid sequence which is at least 60% identical to SEQ ID NO:
 4. 8. The mutant cytochrome protein according to claim 1, wherein said mutant cytochrome protein consists of the amino acid sequence selected from the group consisting of: a) the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4, b) the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, c) an amino acid sequence which is the same as the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4 except that one or several amino acids are substituted, deleted, inserted, and/or added with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained, and d) an amino acid sequence which is the same as the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4 except that one or several amino acids are substituted, deleted, inserted, and/or added with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained. e) an amino acid sequence with a sequence identity of not less than 80% to the amino acid sequence consisting of amino acids 314 to 425 of SEQ ID NO:4 with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained; and f) an amino acid sequence with a sequence identity of not less than 80% to the amino acid sequence consisting of amino acids 330 to 425 of SEQ ID NO:4 with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained.
 9. The mutant cytochrome protein according to claim 1, wherein said mutant cytochrome protein consists of the amino acid sequence selected from the group consisting of: a) the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4, b) the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4, c) an amino acid sequence which is at least 90% identical to the amino acid sequence of amino acids 314 to 425 of SEQ ID NO:4 with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained, and d) an amino acid sequence which is at least 90% identical to the amino acid sequence of amino acids 330 to 425 of SEQ ID NO:4 with the proviso that the third heme-binding domain of amino acids 334 to 338 is maintained.
 10. A DNA encoding the mutant cytochrome protein according to claim
 1. 11. A recombinant vector comprising the DNA according to claim
 10. 12. A cell transformed with the recombinant vector according to claim
 11. 13. An oxidoreductase-cytochrome complex comprising the mutant cytochrome protein according to claim 1 and a catalytic subunit protein of an oxidoreductase.
 14. The oxidoreductase-cytochrome complex according to claim 13, wherein the oxidoreductase is glucose dehydrogenase.
 15. A biosensor comprising an enzyme electrode comprising the oxidoreductase-cytochrome complex according to claim
 13. 16. A method for measuring the concentration of a target substance in a sample, comprising adding the sample to the biosensor according to claim 15, applying an electric potential to the enzyme electrode, measuring the response current, and then calculating the concentration of the target substance contained in the sample based on the response current.
 17. The method according to claim 16, wherein an electric potential of 0 to +300 mV is applied to the enzyme electrode with respect to a silver-silver chloride electrode. 